Curcumin induces stabilization of Nrf2 protein through Keap1 cysteine modification.

The present study was aimed to investigate the effects of curcumin, a representative chemopreventive phytochemical with pronounced antioxidant and anti-inflammatory properties, on activation of Nrf2 and expression of its target protein heme oxygenase-1 (HO-1) in mouse skin in vivo and in cultured murine epidermal cells. Treatment of mouse epidermal JB6 cells with curcumin resulted in the induction of HO-1 expression, and this was abrogated in cells transiently transfected with Nrf2 siRNA. While curcumin treatment increased protein expression of Nrf2, it did not alter the steady-state level of the Nrf2 mRNA transcript. Treatment of cells with curcumin stabilized Nrf2 by inhibiting ubiquitination and subsequent 26S proteasomal degradation of this transcription factor. Tetrahydrocurcumin, a non-electrophilic analogue of curcumin that lacks the α,β-unsaturated carbonyl group, failed to induce HO-1 expression as well as nuclear translocation of Nrf2 and its binding to the antioxidant/electrophile response elements. Cells transfected with a mutant Keap1 protein in which cysteine 151 (Cys151) is replaced by serine exhibited marked reduction in curcumin-induced Nrf2 transactivation. Mass spectrometric analysis revealed that curcumin binds to Keap1 Cys151, supporting that this amino acid is a critical target for curcumin modification of Keap1, which facilitates the liberation of Nrf2. Thus, it is likely that the α,β-unsaturated carbonyl moiety of curcumin is essential for its binding to Keap1 and stabilization of Nrf2 by hampering ubiquitination and proteasomal degradation.

Shin JW ，Chun KS ，Kim DH ，Kim SJ ，Kim SH ，Cho NC ，Na HK ，Surh YJ ... -  《-》

17-Oxo-docosahexaenoic acid induces Nrf2-mediated expression of heme oxygenase-1 in mouse skin in vivo and in cultured murine epidermal cells.

Recently, growing attention has been given to new classes of bioactive lipid mediators derived from ω-3 polyunsaturated fatty acids, such as docosahexaenoic acid (DHA), especially in the context of their role as endogenous signal modulators. One such molecule is 17-oxo-DHA, generated from DHA by the action of COX2 and a dehydrogenase. The redox-sensitive transcription factor, Nrf2 plays a key role in cellular stress responses. In the present study, the effects of 17-oxo-DHA on Nrf2-mediated expression of cytoprotective enzymes were examined in mouse skin in vivo and cultured murine epidermal JB6 cells. Topical application of 17-oxo-DHA markedly elevated the nuclear localization of Nrf2 and expression of heme oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase-1 in hairless mouse skin. In contrast to 17-oxo-DHA, the non-electrophilic metabolic precursor 17-hydroxy-DHA was a much weaker inducer of Nrf2 activation and its target protein expression. Likewise, 17-oxo-DHA significantly enhanced nuclear translocation and transcriptional activity of Nrf2 with concomitant upregulation of HO-1 expression in cultured JB6 cells. 17-Oxo-DHA was a much stronger inducer of Nrf2-mediated antioxidant response than its parent molecule, DHA. HO-1 expression was abolished in Nrf2 knockdown JB6 cells or embryo fibroblasts from Nrf2 knock out mice. 17-Oxo-DHA also markedly reduced the level of Keap1 protein by inducing ubiquitination. Mutation of Cys151 and Cys273 in Keap1 abrogated 17-oxo-DHA-induced ubiquitination and proteasome-mediated degradation of Keap1 as well as HO-1 expression, suggesting that these cysteine residues are putative sites for 17-oxo-DHA binding. Further, Keap1 degradation stimulated by 17-oxo-DHA coincided with accumulation of the autophagy substrate, p62/SQSTM1.

Jamil MU ，Kim J ，Yum HW ，Kim SH ，Kim SJ ，Kim DH ，Cho NC ，Na HK ，Surh YJ ... -  《-》

Withaferin A induces heme oxygenase (HO-1) expression in endothelial cells via activation of the Keap1/Nrf2 pathway.

Withaferin A (WA), a natural phytochemical derived from the plant Withania somnifera, is a well-studied bioactive compound exerting a broad spectrum of health promoting effects. To gain better insight in the potential therapeutic capacity of WA, we evaluated the transcriptional effects of WA on primary human umbilical vein endothelial cells (HUVECs) and an endothelial cell line (EA.hy926). RNA microarray analysis of WA treated HUVEC cells demonstrated increased expression of the antioxidant gene heme oxygenase (HO-1). Transcriptional regulation of this gene is strongly dependent on the transcription factor NF-E2-related factor 2 (Nrf2), which senses chemical changes in the cell and coordinates transcriptional responses to maintain chemical homeostasis via expression of antioxidant genes and cytoprotective Phase II detoxifying enzymes. Under normal conditions, Nrf2 is kept in the cytoplasm by Kelch-like ECH-associated protein 1 (Keap1), an adaptor protein controlling the half-life of Nrf2 via constant proteasomal degradation. In this study we demonstrate that WA time- and concentration-dependently induces HO-1 expression in endothelial cells via upregulation and increased nuclear translocation of Nrf2. According to the crucial negative regulatory role of Keap1 in Nrf2 expression levels, a direct interaction of WA with Keap1 could be demonstrated. In vitro and in silico evaluations suggest that specific cysteine residues in Keap1 might be involved in the interaction with WA.

Heyninck K ，Sabbe L ，Chirumamilla CS ，Szarc Vel Szic K ，Vander Veken P ，Lemmens KJA ，Lahtela-Kakkonen M ，Naulaerts S ，Op de Beeck K ，Laukens K ，Van Camp G ，Weseler AR ，Bast A ，Haenen GRMM ，Haegeman G ，Vanden Berghe W ... -  《-》

Piceatannol induces heme oxygenase-1 expression in human mammary epithelial cells through activation of ARE-driven Nrf2 signaling.

Piceatannol (3,4,3',5'-tetrahydroxy-trans-stilbene), derived from the seeds of Euphorbia lagascae, has been reported to have anti-proliferative, anti-inflammatory, and antioxidant properties. However, the mechanisms underlying its chemoprotective effects remain largely unresolved. In the present study, we found that piceatannol treatment (30 microM) significantly upregulated the expression of the antioxidant enzyme heme oxygenase-1 (HO-1) and its mRNA transcript at 6h and 3h, respectively in human breast epithelial (MCF10A) cells. A redox-sensitive transcription factor NF-E2-related factor (Nrf2) plays a pivotal role in induced expression of many cytoprotective enzymes including HO-1. Piceatannol induced translocation of Nrf2 into the nucleus and its transcriptional activities when treated to the MCF10A cells for 6h. Upregulation of HO-1 expression by piceatannol through direct binding of Nrf2 to antioxidant response element (ARE) was verified by the chromatin immunoprecipitation (ChIP) assay. siRNA knock down of Nrf2 gene abolished piceatannol-induced HO-1 expression. In addition, piceatannol-induced activation of Nrf2 and/or HO-1 expression was abrogated by the pharmacological inhibitor (LY294002) as well as the kinase-dead form of Akt. In an attempt to elucidate the molecular mechanisms underlying cytoprotective or chemoprotective activity exerted by piceatannol, we examined its effect on the signaling pathways responsible for induction of HO-1 expression. We hypothesize that an electrophilic quinone formed as a consequence of oxidation of piceatannol bearing the catechol moiety may bind directly to Kelch-like ECH-associated protein 1 (Keap1), an inhibitory protein that sequesters Nrf2 in the cytoplasm. This will diminish the affinity of Keap1 for Nrf2. The thiol reducing agents, dithiothreitol (100 microM) or beta-mercaptoethanol (1.4 microM), attenuated piceatannol-induced Nrf2 activation and HO-1 expression. It is hence likely that piceatannol modifies specific cysteine residues of Keap1, which allows Nrf2 to translocate into the nucleus and bind to ARE, leading to enhancement of the expression of HO-1. The characteristic catechol moiety of piceatannol appears to be critical for induction of Nrf2 activation and subsequent upregulation of HO-1.

Lee HH ，Park SA ，Almazari I ，Kim EH ，Na HK ，Surh YJ ... -  《-》

Zerumbone induces heme oxygenase-1 expression in mouse skin and cultured murine epidermal cells through activation of Nrf2.

Shin JW ，Ohnishi K ，Murakami A ，Lee JS ，Kundu JK ，Na HK ，Ohigashi H ，Surh YJ ... -  《-》